The multi-chain immune recognition receptors (MIRR) which mediate cell activation in the immune response include T cell receptors for antigen, B cell receptors, and Fc receptors. The structural basis for MIRR function at the plasma membrane will be investigated, using as a paradigm the high affinity Fc receptor (FcepsilonRI) for immunoglobulin E (IgE) which plays a central role in the allergic immune response. Proposed studies will focus on plasma membrane heterogeneity, dynamics and structure as these are involved in IgE-FcepsilonRI signaling, and in particular on the role liquid-ordered, detergent-resistant regions play in the initial coupling between FcepsilonRI and Lyn tyrosine kinase. Specific aim 1 will examine features of FcepsilonRI structure that are critical for its interaction with detergent resistant membranes, using several different chimeric receptors, together with site-specific mutagenesis and cholesterol photoaffinity labeling studies. Specific aim 2 will apply advanced biophysical methods including quantitative fluorescence microscopy, electron spin resonance and mass spectrometry to characterize these cholesterol-dependent, detergent-resistant regions in plasma membranes of whole cells and sub-cellular preparations; structural changes that occur as a result of cell activation will be investigated. For example, real time interactions between Lyn anchored to the inner leaflet of the plasma membrane and antigen-aggregated FcepsilonRI will be monitored on intact cells using green fluorescent protein-conjugated analogues to detect proximity changes with fluorescence resonance energy transfer and mobility changes with fluorescence correlation spectroscopy and imaging. The structural basis for the interaction of F- actin with the plasma membrane and its regulation of FcepsilonRI signaling will also be investigated. These studies will test te general hypothesis that structural organization at the plasma membrane is important for receptor-mediated signaling in the immune response.